Herpes simplex virus (HSV) contains a double-stranded, linear DNA genome comprised of approximately 152 kbp of nucleotide sequence, which encodes more than 80 genes. The viral genes are transcribed by cellular RNA polymerase II and are temporally regulated, resulting in the transcription and subsequent synthesis of gene products in roughly three discernable phases. These phases, or kinetic classes of genes are referred to as the Immediate Early (IE, or .alpha.), Early (E, or .beta.) and Late (L, or .gamma.) genes. Immediately following the arrival of the genome of a virus in the nucleus of a newly infected cell, the IE genes are transcribed. The efficient expression of these genes does not require prior viral protein synthesis. The products of IE genes are required to activate transcription and regulate the remainder of the viral genome.
One IE protein, Infected Cell Polypeptide 4 (ICP4), also known as .alpha.4, or Vmw175, is absolutely required for both virus infectivity and the transition from IE to later transcription. Owing to its complex, multifunctional nature and its central role in the regulation of HSV gene expression, ICP4 has been the subject of numerous genetic and biochemical studies. (See DeLuca, et al., 1987, Nucleic Acids Res. 15:4491-4511; DeLuca, et al., 1988, J. Virol. 62:732-743; Paterson, et al., 1988, Virology, 166:186-196; Paterson, et al., 1988, Nucleic Acids Res. 16:11005-11025; Shepard, et al., 1989, J. Virol. 63:3714-3728; and Shepard, et al., 1991, J. Virol. 65:787-795). Aiding in these studies was the development of a system to grow herpes viruses that contain mutations which inactivate essential viral proteins. In this case, cell lines were generated by cotransformation with a plasmid DNA that encoded the neomycin resistance gene from E. coli under the control of SV40 early promoter and a plasmid encoding the wild-type ICP4gene. (See DeLuca, et al., 1985, J. Virol. 56:558-570 and DeLuca, et al., 1987, Nucleic Acids Res. 15:4491-4511). These stable cell lines were used to generate and propagate mutant viruses that are void of ICP4 activity. (See DeLuca, et al., 1985, J. Virol. 56:558-570; DeLuca, et al., 1988, J.Virol. 62:732-743; Imbalzano, et al. 1991, J. Virol. 65:565-574; Shepard, et al., 1989, J. Virol., 63:3714-3728, 1989; and Shepard, et al., 1991, J. Virol. 65:787-795). Since the first report of this approach to HSV genetics, numerous studies have followed utilizing this strategy.
From the phenotype of viruses deleted in ICP4, it became evident that such viruses would be potentially useful for gene transfer purposes. Several studies have been published exploring the potential use of such viruses for gene transfer. (See Breakefield, et al., 1991, Treatment of Genetic Diseases, Churchill Livingstone, Inc.; and Chocca, et al., 1990, The New Biologist 2:739-746). One property of viruses deleted for ICP4 that makes them desirable for gene transfer is that they only express the five other IE genes: ICP0, ICP6, ICP27, ICP22 and ICP47. (See DeLuca, et al., 1985, J. Virol. 56:558-570). This excludes the expression of viral genes encoding proteins that direct viral DNA synthesis, as well as the structural proteins of the virus. This is desirable from the standpoint of minimizing possible deleterious effects on host cell metabolism following gene transfer.
Despite the fact viruses deleted for ICP4 are blocked at the earliest stage of infection genetically possible subsequent to the delivery of the genome to the host cell nucleus, two phenomena have complicated the use of such viruses for effective gene transfer, or therapy. First, viruses deleted for essential genes, such as ICP4-deficient viruses, require that they are propagated on cultured cells engineered to contain and express the gene deleted from the virus. (See DeLuca, N. A., 1985, J. Virol. 56:558-570). This often results in a subpopulation of viruses that are no longer deleted for that gene due to homologous recombination events between the mutant viral genome and the wild-type gene resident in the host cell genome. (See DeLuca, et al., 1985. J. Virol. 56:558-570). In some cases, this is minimized by deleting from the virus HSV sequences flanking the deleted gene and excluding these sequences from the plasmid used to generate the permissive transformed cell line. Therefore, the gene resident in the transformed cell line does not have flanking nucleotide sequence homology on both sides to promote homologous recombination. This is the case for the ICP4 deletion virus-transformed cell line pair, d120-E5 cells (See DeLuca, et al., 1985, J. Virol. 56:558-570 and DeLuca, et al., 1987, Nucleic Acids Res. 15:4491-4511) and the ICP27 deletion virus-transformed cell line pair, 5 dl 1.2-2-3 cells. (See McCarthy, et al., 1989. J. Virol. 63:18-27).
Secondly, despite only expressing the four other immediate early proteins, ICP4-deficient viruses are toxic to cells in culture and presumably to the majority of cells in an animal. This is most probably due to the expression of one or more of the remaining immediate early proteins and not primarily due to components of the incoming capsid since certain defective HSV virus particles, which contain all the capsid components and none of the IE genes, are not toxic. In addition, ICP4 deficient viruses shutoff host cell protein synthesis through the activity of the UL41 virion gene product (Leib, et al., 1989, J. Virol. 63:759-768; Read, et al., 1993, J. Virol. 67:7149-7160.) The cytotoxicity of ICP4.sup.(-) HSV strains is not due to ICP22 and ICP47 and is probably mediated by multiple gene products (Johnson, et al., 1992, J. Virol. 66:2952-2965.)
An HSV mutant virus deficient for the non-essential UL41 gene product is described by Read, et al. (1993, J. Virol. 67:7149-7160.) No potential use of this UL41.sup.(-) HSV mutant as a eukaryotic gene transfer vehicle is suggested by the authors.
HSV ribounucleotide reductase consists of a large (ICP6) and small subunit. Goldstein and Weller (1988, J. Virol. 62:196-205) disclose (1) ribonucleotide reductase activity is not essential for HSV growth, and (2) ICP6may be inactivated via homologous recombination with a reporter gene (lacZ). The resulting ICP6.sup.(-) :LacZ.sup.(+) HSV strain expresses lacZ and is not dependent upon exogenous ribonucleotide reductase for viral growth. No potential use of this recombinant HSV strain as a gene therapy vehicle is disclosed, taught or suggested.
Therefore, despite attempts to alleviate various problems with use of known HSV mutant strains upon host cell infection, a need exists for defective herpes simplex virus strains that exhibit efficient growth in a controlled laboratory complementing system, a lower level of wild-type virus regeneration and lowered cytotoxic effects.